phytotaxa 233

Phytotaxa 233 (1): 001–026www.mapress.com/phytotaxa/ Copyright © 2015 Magnolia Press Article PHYTOTAXA

ISSN 1179-3155 (print edition)

ISSN 1179-3163 (online edition)

Accepted by Jian-Kui Liu: 25 Sept. 2015; published: 30 Oct. 2015

http://dx.doi.org/10.11646/phytotaxa.233.1.1

1Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0

Botryosphaeriaceae associated with Tectona grandis (teak) in Northern Thailand

MINGKWAN DOILOM1,2, LUCAS A. SHUTTLEWORTH3, JOLANDA ROUX3, EKACHAI CHUKEATIROTE1,2 & KEVIN D. HYDE1,2,4,5,* 1Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand2School of Science, Mae Fah Luang University, Chiang Rai. 57100, Thailand3Department of Plant Sciences, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Preto-ria, 0028, South Africa4Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, China5World Agroforestry Centre, East and Central Asia, Kunming 650201, Yunnan, China *Correspondence: [email protected]

Abstract

Tectona grandis (teak) is one of the most important economic timbers worldwide. Limited studies exist on the potential pathogens of these trees. Fungi in the Botryosphaeriaceae are cosmopolitan opportunistic pathogens, endophytes and sap-robes of numerous hosts. Both symptomatic and asymptomatic branch and stem sections, as well as leaves were collected from T. grandis in plantations and forests in four provinces of northern Thailand with the aim of identifying species of Botryosphaeriaceae associated with these trees. Morphology and multi-locus phylogenies (ITS, TEF1-α, β-tubulin) were used to identify the Botryosphaeriaceae species. Six species from four different genera were found on T. grandis in Northern Thailand. These included Dothiorella tectonae sp. nov., Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae, Pseudofusicoccum adansoniae and Sphaeropsis eucalypticola. Dothiorella tectonae is introduced here as a novel species and compared with other species in the genus. Dothiorella tectonae, L. brasiliense, L. pseudotheobromae, L. theobromae, P. adansoniae and S. eucalypticola are first reports for T. grandis in Thailand. Variations in morphology between descriptions of previously described species and that obtained in this study are described to facilitate future identification of species.

Key words: Dothiorella, Lasiodiplodia, Pseudofusicoccum, Sphaeropsis, multi-gene phylogenetics, taxonomy

Introduction

Tectona grandis (teak) is one of the most economically valuable tropical hardwood trees globally. Natural teak forests occur in only four countries in the world including Burma, India, Lao People’s Democratic Republic and Thailand (Kollert and Cherubini 2012; Thulasidas 2014). India, Lao PDR and Thailand have bans on logging of native teak forests and the export of native teak, and therefore rely on plantations for teak production (Kollert and Cherubini 2012). In 2010, teak plantations in Thailand were reported to cover an area of ~128 000 ha (Kollert and Cherubini 2012). Large areas in northern Thailand are planted with teak plantations and covered by natural teak forests (Graudal et al. 1999). In Chiang Rai Province, the area under productive teak plantations is ~3 321 ha with the income from teak and teak timber products reported as ~120 725 USD (80 USD/m3/year) (Elmagboul et al. undated). Teak has been an important trading commodity in Thailand for over 700 years (Areeya 1992). The timber has a wide range of uses including flooring, decking, framing, bargeboards, carvings, and furniture. It is also used for shipbuilding due to its resistance to sun, heat, cold, rain and seawater (Rondon et al. 1998). Various fungal pathogens are reported to affect teak, including Armillaria mellea and Phellinus noxius causing root rot (Mohd Farid et al. 2005, Owusu 2011), Erythricium salmonicolor causing pink disease (stem cankers and girdling) (Akrof et al. 2014) and Olivea tectonae causing leaf rust (Daly et al. 2006, Pérez et al. 2008, Cabral et al. 2010). Numerous endophytic fungi have been reported from teak leaves in Thailand. These include species of Alternaria, Colletotrichum, Daldinia eschscholtzii, Fusarium, Nigrospora, Penicillium, Phomopsis, and Schizophyllum commune and members of the Xylariaceae (Mekkamol et al. 1997, Mekkamol 1998, Chareprasert et al. 2006). Several

DOILOM ET AL.2 • Phytotaxa 233 (1) © 2015 Magnolia Press

lignicolous marine fungi have been reported on submerged teak blocks, namely Arthrobotrys sp., Byssochlamys fulva, Cylindrocephalum sp., Cytospora sp. and Monodictys pelagica (Vrijimoed et al. 1982, 1986). Most recently, a novel species of Botryosphaeriaceae, Barriopsis tectonae, was described from teak in northern Thailand (Doilom et al. 2014). The Botryosphaeriaceae are a cosmopolitan group of fungi containing pathogens, endophytes and saprobes of mainly woody plant hosts (Crous et al. 2004, Slippers & Wingfield 2007, Trakunyingcharoen et al. 2014, Mehl et al. 2013). The family currently consists of seventeen genera, defined over the last few years based on multi-gene phylogenetic analyses and morphology (Phillips et al. 2013, Slippers et al. 2013). Several species in the Botryosphaeriaceae are important pathogens of economically important crops, including commercial plantation tree species. For example, Diplodia sapinea is an important pathogen of plantation grown Pinus species in the Southern Hemisphere (Smith et al. 2001, Burgess et al. 2004, Reay et al. 2006), Lasiodiplodia theobromae causes die-back and wood discoloration in various parts of the tropics, such as on Acacia mangium in Indonesia (Slippers & Wingfield 2007) and Neofusicoccum species cause disease of Eucalyptus trees grown in plantations in the Southern Hemisphere (Smith et al. 2001). However, virtually no information exists for the Botryosphaeriaceae on T. grandis. The aim of this study was to identify species of Botryosphaeriaceae occurring on T. grandis in northern Thailand. Isolates were collected from both natural forests and plantations of teak. Morphology and multigene sequence analyses were used to identify the isolates obtained from teak.

Materials and Methods

Field collection and fungal isolationsPlant materials were randomly collected from Tectona grandis trees from 35 sites in four provinces in northern Thailand (Chiang Rai, Chiang Mai, Phayao, Phrae Provinces) during the period 2011–2013. Sites for collection were selected to include both teak in natural forests (15 sites) and teak plantations (20 sites). At each site trees were examined for the presence of leaf spots, branch die-back and stem cankers, typical of those caused by species of Botryosphaeriaceae. Saprobes on dead twigs and dead branches were also collected. Furthermore, disease free branches and leaves were collected to isolate endophytic Botryosphaeriaceae from ten plantation sites in Chiang Rai Province. Samples from each tree were placed into separate brown paper bags that were then sealed in larger Zip-lock bags to retain moisture. Samples were refrigerated at 4°C until they could be processed for fungal determination and isolation. In the laboratory, all materials showing disease symptoms were cut into small pieces (1×1 cm.) and surface disinfected in 10% sodium hypochlorite (NaOCl) for 3–5 min, then washed two times with sterile distilled water (1 min each) and blotted dry on sterile filter paper. Disinfected plant tissues were placed on water agar (WA) with streptomycin (0.02 %). Plates were incubated at 25°C for 2–4 days until the onset of fungal growth. Cultures were purified by transferring single hyphal tips and maintained on malt extract agar. For the isolation of possible endophytic Botryosphaeriaceae, disease free leaves (petioles, leaf blades, midribs) and branch samples were cut into 1cm3 sections. These sections were surface disinfested and processed according to Pérez et al. (2010). Colonies resembling Botryosphaeriaceae (fluffy, fast-growing, white colonies turning olive green–grey to black over a few days) were subcultured on to fresh 2% malt extract agar (MEA) and incubated at 25°C for 2–4 days. Leaves with leaf spots containing fruiting bodies and fruiting bodies on dead branches and twigs, resembling those of species in the Botryosphaeriaceae, were isolated using the single spore technique described by Chomnunti et al. (2014). Representative cultures were prepared for analyses using DNA sequencing and morphological analyses and for deposition in recognized culture collections. Type herbarium material was deposited in the herbarium of Mae Fah Luang University, Chiang Rai, Thailand (MFLU). Representative copies of all species obtained in this study have also been deposited in the culture collections of the Mae Fah Luang Culture Collection, Thailand (MFLUCC) with duplicates stored at Mycothèque de l’Université catholique de Louvain, Belgium (MUCL).

DNA extraction, PCR amplification and sequencingTwelve representative isolates were selected for the molecular analyses (Table 1) Genomic DNA was extracted directly from actively growing mycelia using PrepMan™ Ultra (Applied Biosystems, Foster City, CA, U.S.A.) following the

BOTRYOSPHAERIACEAE IN NORTHERN THAILAND Phytotaxa 233 (1) © 2015 Magnolia Press • 3

manufacturer’s protocol. The internal transcribed spacer regions 1 and 2 (ITS), including 5.8S region fragments of the protein coding genes, translation elongation factor 1-alpha (TEF1-α) and β-tubulin (BT), were used for identification of isolates. The ITS regions were amplified and sequenced with the primers ITS1/ITS4 (White et al. 1990), TEF1-α with EF1F/EF2R (Jacobs et al. 2004) and EF688F/EF1251R (Alves et al. 2008) for Lasiodiplodia isolates, and the BT region with the Bt2a and Bt2b primers (Glass & Donaldson 1995). PCR reactions were performed in 25 μL final volumes and consisted of 5 μL 5x MyTaq Reaction Buffer (Bioline, U.S.A.), 0.15 μL MyTaq™ DNA Polymerase (Bioline, U.S.A.), 0.5 μL of each primer (10 mM stock) (Whitehead Scientific, Cape Town, South Africa), and 1 μL of the PrepMan DNA extract. Final reaction volumes were adjusted to 25 μL by adding sterile Sabax water (Adcock Ingram Critical Care, Johannesburg, South Africa). Thermocycling was completed on a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.A.) under the following conditions. Initial denaturation of 5 min at 95 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 52 °C, 1 min at 72 °C and a final extension period of 7 min at 72 °C. The annealing temperatures of the TEF1-α and BT reactions were changed to 55 °C. Amplification was confirmed by staining 2 μL aliquots of PCR products with 3 μL of GelRed™ Nucleic Acid Gel stain (Biotium, Hayward, CA, U.S.A.) and separated on a 1 % agarose gel. Fragments were visualized on a Bio-Rad GelDoc EZ system. PCR products were cleaned by gel filtration using Sephadex G-50 columns (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions. Sequencing PCR reactions were completed according to the method described by Doilom et al. (2014). Products were sequenced at the DNA Sequencing Facility, Bioinformatics and Computational Biology Unit, University of Pretoria, South Africa. Both forward and reverse strands were completed to ensure sequence integrity. Consensus sequences were generated using Geneious® R7 (Biomatters Ltd., New Zealand).

Phylogenetic analysesAll sequences obtained after sequencing were used for BLAST searches in the nucleotide database of GenBank (www http://blast.ncbi.nlm.nih.gov/) to determine their most likely genus identity. This information was used to compile data sets for phylogenetic analyses. Reference sequences used in the phylogenetic analyses were obtained from recent relevant literature and GenBank (Lui et al. 2012, Phillips et al. 2013, Slippers et al. 2013, Hyde et al. 2014; Nilsson et al. 2014). Outgroups for each data set were selected from phylogenetically closely related genera of the Botryosphaeriaceae. These included Spencermartinsia viticola (CBS 117009, CBS 302.75), Diplodia mutila (CBS 112553), D. seriata (CBS 112555), Neofusicoccum parvum (CMW 9081, CBS 110301) and Barriopsis fusca (CBS 174.26). Three individual datasets for each of the loci, ITS, TEF1-α and BT, and one concatenated ITS/TEF1-α/BT dataset were assembled and analysed. Data sets were aligned online using the MAFFT version 7.158 server (http://mafft.cbrc.jp/alignment/ server/). Alignments were checked visually and corrected for alignment errors. Potential conflict among datasets was estimated with a conditional comparison test with maximum parsimony bootstrap (BS) values ≥70% (Kellogg et al. 1996, Mason-Gamer & Kellogg 1996, Johnson & Soltis 1998). The number of polymorphisms in each of the individual marker datasets was also considered when delineating species, employing genealogical concordance phylogenetic species recognition (Taylor et al. 2000). Phylogenetic trees were inferred with maximum parsimony (MP) and Bayesian inference (BI). Maximum parsimony (MP) analyses were conducted using MEGA 6 (Tamura et al. 2013) and PAUP version 4.0 b10 (Swofford 2003). Uninformative characters were excluded. All informative characters were unordered and of equal weight. The heuristic search function was used with 1000 random stepwise addition replicates and tree bisection-reconnection (TBR) as the branch-swapping algorithm. Goodness of fit values, including the consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI), were calculated. Statistical support for branches was estimated using maximum parsimony bootstrap (MPBS) analysis with 1000 replicates (Felsenstein 1985). Phylogenetic trees were visualized and annotated using Treeview (Page 1996) and formatted using PowerPoint 2010 (Microsoft Corporation, WA, U.S.A.). Bayesian inference was performed using the Markov Chain Monte Carlo (MCMC) method with MrBayes v. 3.2.2 (Huelsenbeck & Ronquist 2001). MrModeltest 2.2 (Nylander 2004) was used to select the best-fit nucleotide substitution models under the Akaike information criterion (AIC). Four chains were run for the individual and combined data sets. The MCMC algorithm was started from a random tree topology. Five million generations was selected with a sampling frequency every 100 generations. The Tracer v. 1.6 (Rambaut & Drummond 2003) programme was used to check the effective sampling sizes (ESS), which should be above 200, the stable likelihood plateaus and burn-in value. The first 5,000 generations were excluded as burn-in. Posterior probabilities were viewed with FigTree v1.3.1 (Rambaut 2009).


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3095

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S 12

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2637

So

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Pt. a

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...C

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TA

BL

E 1

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Tax

onC

ultu

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cces

sion

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Cou

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Ex

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6733

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35

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6731

40

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6731

41

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Sp. e

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C 1

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L 55

412

Th

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KM

3969

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5103

65

Sp. p

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TE-U

513

2 Ex

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Sout

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3433

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3130

Sp. v

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186.

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6733

25EU

6732

93EU

6731

28

Sp. v

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BS

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63

Luxe

mbo

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Vi. a

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EU67

3324

EU

6732

92EU

6731

271 A

cron

yms

of c

ultu

re c

olle

ctio

ns: A

TC

C:

Am

eric

an T

ype

Cul

ture

Col

lect

ion,

Virg

inia

, USA

; BL

: B

.T. L

inal

dedd

u, U

nive

rsità

deg

li St

udi d

i Sas

sari,

Ita

ly; B

RIP

: C

ultu

re c

olle

ctio

n, Q

ueen

slan

d D

epar

tmen

t of A

gric

ultu

re an

d Fi

sher

ies,

Que

ensl

and,

Aus

tralia

; CA

P: P

erso

nal c

ultu

re co

llect

ion A

lan

J.L. P

hilli

ps, U

nive

rsid

ade N

ova d

e Lis

boa,

Por

tuga

l; C

BS:

Cen

traal

bure

au v

oor S

chim

mel

cultu

res,

Utre

cht,

The

Net

herla

nds;

CM

M: C

ultu

re C

olle

ctio

n of

Phy

topa

thog

enic

Fun

gi “

Prof

. Mar

ia M

enez

es”,

Uni

vers

idad

e Fe

dera

l Rur

al d

e Pe

rnam

buco

, Rec

ife, B

razi

l; C

MW

: Tre

e Pa

thol

ogy

Coo

pera

tive

Prog

ram

, For

estry

and

Agr

icul

tura

l Bio

tech

nolo

gy I

nstit

ute,

Uni

vers

ity o

f Pr

etor

ia, S

outh

Afr

ica;

CPC

: C

ultu

re c

olle

ctio

n of

P.W

. Cro

us h

ouse

d at

CB

S; D

AR

: Pl

ant P

atho

logy

Her

bariu

m, O

rang

e A

gric

ultu

ral I

nstit

ute,

DPI

, Ora

nge,

NSW

, Aus

tralia

; IC

MP:

Inte

rnat

iona

l Col

lect

ion

of M

icro

orga

nism

s fr

om P

lant

s, La

ndca

re R

esea

rch,

New

Zea

land

; IM

I: In

tern

atio

nal M

ycol

ogic

al In

stitu

te, C

BI-

Bio

scie

nce,

Egh

am, B

akeh

am L

ane,

UK

; IR

AN

: Ir

ania

n Fu

ngal

Cul

ture

Col

lect

ion,

Ira

nian

Res

earc

h In

stitu

te o

f Pl

ant P

rote

ctio

n, I

ran;

MFL

UC

C:

Mae

Fah

Lua

ng U

nive

rsity

Cul

ture

Col

lect

ion,

C

hian

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Morphological characterization of isolatesMorphological characterization of the twelve isolates was carried out according to the methods described in Doilom et al. (2013, 2014). Fungal structures were stained using Lactophenol Cotton Blue (LPCB). Growth rates were determined using a five mm diameter cork borer to transfer mycelium plugs to 2% MEA. Five replicates of each isolate were made and kept at 5, 15, 25 and 30 °C for new taxa. All fungal taxa were kept in the incubator in the dark at 25 °C for one week. Culture studies were completed from isolates grown at 25 °C for one week. Colony colour was then determined with the Methuen handbook of colour (Kornerup &Wanscher 1967).

Results and Discussion

Fungal isolates

Isolates resembling species of Botryosphaeriaceae were obtained from 95 isolates /12 sites. One representative isolate per site, for each colony morphotype was used for identification using DNA sequence data.

Phylogenetic analyses

Based on MP analyses of ITS data including all genera in the Botryosphaeriaceae, four genera of Botryosphaeriaceae were identified namely Dothiorella (1 isolate), Lasiodiplodia (7 isolates), Pseudofusicoccum (3 isolates) and Sphaeropsis (1 isolate) (data not shown). The datasets of each genus were then separately analysed for species identification using individual ITS, TEF1-α and BT, as well as combined phylogenies. Alignment details and nucleotide substitution models for the individual ITS, TEF1-α, BT and combined datasets are provided in Table 2. Dothiorella group: In the ITS MP phylogeny the Dothiorella isolate (MFLUCC 12-0382) obtained from T. grandis in this study grouped sister to, but distinct from Do. uruguayensis (CBS 124908) and Do. striata (ICMP 16819 and ICMP 16824) (Figure 1a). For BI phylogeny of ITS it grouped as distinct linear (Figure 1b). In the TEF1-α and BT MP trees it grouped closest to Do. brevicollis (CBS 130411 and CBS 130412) and Do. longicollis (CBS 122067 (unavailable for BT sequence data in GenBank) and CBS 122068). In the TEF1-α analyses it was strongly supported as a distinct species (MPBS 95, PP 0.90) (Figure 1c), but in the BT tree the branch was not strongly supported for MP and PP (Figure 1d). However, resolution of species complexes of Botryosphaeriaceae can be better achieved by using combined ITS and TEF1-α gene analyses (Liu et al. 2012, Phillips et al. 2013, Abdollahzadeh et al. 2014). Dothiorella cannot be distinguished from Spencermartinsia using ITS sequence data alone. It is, therefore, essential to combine ITS with TEF1-α or other protein coding genes for delineating the identity of these isolates (Phillips et al. 2013). In the combined ITS/TEF1-α/BT MP and PP phylogenies the isolate from T. grandis grouped closest to, but distinct from Do. brevicollis and Do. longicollis, (MPBS 93% and PP 1.00), suggesting that it represents a novel species (Figure 1e). BI trees of individual TEF1-α and BT genes and combined genes had similar topologies to the MP trees. The Dothiorella sp. from T. grandis showed multiple nucleotide differences between itself and closely related taxa for all gene regions analysed (Table 3). In the ITS region it differed from Do. brevicollis with 10 polymorphisms, 15 compared to Do. longicollis, 17 compared to Do. striata and 13 when compared to Do. uruguayensis. In the TEF1-α it differed from Do. brevicollis with 30 polymorphisms, 31, 51 and 57 compared to Do. longicollis, Do. striata and Do. Uruguayensis respectively. In the BT it differed with 12, 13 and 16 from Do. brevicollis, Do. longicollis, and Do. striata. BT sequence data for Do. longicollis and Do. uruguayensis was not available, therefore, comparisons could not be made. These differences clearly show that this isolate represents a novel species of Dothiorella and it is described below at Do. tectonae. Lasiodiplodia group: Three species of Lasiodiplodia were obtained (Figure 2). Isolate MFLUCC 12-0293 grouped with L. theobromae in the individual (ITS, TEF1-α, BT) and combined analyses, but with weak bootstrap support for MP and PP. In the combined phylogeny MPBS of 64% and PP of 0.94 was obtained. Isolate MFLUCC 11-0414 grouped within L. brasiliense (MPBS = 95%, PP = 1.00 in the combined phylogeny). Individual gene trees for ITS, TEF1-α and BT analyses showed five isolates (MFLUCC 12-0053, MFLUCC 12-0294, MFLUCC 12-0295, MFLUCC 12-0772, MFLUCC 12-0796) grouped with L. pseudotheobromae with poor bootstrap support for MP and PP, but in the combined phylogeny they strong bootstrap support (MPBS=99%, PP =1.00) was obtained. BI trees of individual genes and combined genes had similar topologies to the MP trees. Pseudofusicoccum group: Strains MFLUCC 13-0705, MFLUCC 14-0516 and MFLUCC 14-0517 grouped with


Ps. adansoniae (ex-type culture) in the individual ITS and TEF1-α phylogenies (MPBS: ITS = 93%, TEF1-α = 91%, PP: ITS = 0.66, TEF1-α = 0.77), as well as in the combined ITS and TEF1-α phylogenies (MPBS=100%, PP =0.93) and are therefore considered as this species (Figure 3). Individual gene trees for ITS and TEF1-α analysis gave a similar topology to the combined analyses and is not shown. BI trees of individual genes and combined genes had similar topologies to the MP tree. There are few BT sequences from Pseudofusicoccum species in GenBank and thus, individual BT analysis was not analyzed in this study.

FIGURE 1. Phylogenetic trees resulting from individual analyses of each dataset and combined dataset. a. One of >200 most parsimonious trees (CI= 0.622, HI= 0.378, RI= 0.863, RC= 0.537) resulting from ITS analysis for 40 taxa in Dothiorella species, b. Bayesian analysis tree resulting from ITS analysis, c. One of >200 most parsimonious trees (CI= 0.635, HI= 0.365, RI= 0.867, RC= 0.550) resulting from TEF1-α analysis for 39 taxa, d. One of 24 most parsimonious trees (CI= 0.750, HI= 0.250, RI= 0.891, RC= 0.668) resulting from BT analysis for 29 taxa, e. One of 63 most parsimonious trees (CI= 0.642, HI= 0.358, RI= 0.863, RC= 0.554) resulting from combined ITS, TEF1-α and BT analysis for 40 taxa. The tree is rooted to two isolates of Spencermartinsia viticola. Maximum parsimony bootstrap values ≥50%, Bayesian posterior probabilities ≥ 0.90 (MPBS/PP) are given at the nodes. Type isolates are marked with T. Isolates from this study are in blue bold.


FIGURE 2. One of 48 most parsimonious trees (CI= 0.674, HI= 0.326, RI= 0.881, RC= 0.594) resulting from a combined ITS, TEF1-α and BT analysis for 56 taxa in Lasiodiplodia species. The tree is rooted to isolates of Diplodia mutila and D. seriata. Maximum parsimony bootstrap values ≥50%, Bayesian posterior probabilities ≥ 0.90 (MPBS/PP) are given at the nodes. Type isolates are marked with T. Isolates from this study are in bold.


FIGURE 3. One of 12 most parsimonious trees (CI= 0.909, HI= 0.091, RI= 0.943, RC= 0.857) resulting from combined ITS and TEF1-α analysis for 20 taxa in Pseudofusicoccum species. The tree is rooted to two isolates of Neofusicoccum parvum. Maximum parsimony bootstrap values ≥50%, Bayesian posterior probabilities ≥ 0.90 (MPBS/PP) are given at the nodes. Type isolates are marked with T. Isolates from this study are in bold.

Sphaeropsis group: Isolate MFLUCC 13-0701 grouped with Sp. eucalypticola (ex-type culture) in the individual ITS, TEF1-α and BT phylogenies (MPBS: ITS = 86%, TEF1-α = 100%, BT = 98%, PP: ITS = 0.99, TEF1-α = 1.00, BT = 0.99) (tree not shown), and also grouped with Sp. eucalypticola (MFLUCC11-0654 and MFLUCC11-0579) in the combined phylogeny with MPBS (100%) and PP (1.00) and is therefore considered to be Sp. eucalypticola (Figure 4). Individual gene trees of MP and BI trees for ITS, TEF1-α and BT analysis was similar in topology to trees generated from combined analyses and is not shown. The BI tree of combined analyses was similar in topology and clades to the MP tree. Several studies have confirmed that single gene regions are insufficient to delimit cryptic species in genera of Botryosphaeriaceae (de Wet et al. 2003, Slippers et al .2004a, b). The combined gene analyses are required to resolve species boundaries in the genera (Alves et al. 2008, Abdollahzadeh et al. 2010). Therefore, our final phylogenetic analyses are based on the combination of multi-locus phylogenies (ITS, TEF1-α, and/or β-tubulin). These result identified six species from four different genera (Dothiorella tectonae sp. nov., Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae, Pseudofusicoccum adansoniae and Sphaeropsis eucalypticola) of Botryosphaeriaceae associated with T. grandis in four provinces in northern Thailand (Figures 1–4).


FIGURE 4. One of 4 most parsimonious trees (CI= 0.912, HI= 0.088, RI= 0.927, RC= 0.845) resulting from combined ITS, TEF1-α and BT analysis for nine taxa in Sphaeropsis species. The tree is rooted to Barriopsis fusca. Maximum parsimony bootstrap values ≥70%, Bayesian posterior probabilities ≥ 0.90 are given at the nodes (MPBS/PP). Type isolates are marked with T. Isolates from this study are in bold.


TABLE 2. Alignment length, parsimony analyses output and nucleotide substitution models used in the phylogenetic analyses.Genus Dataset Number of

ingroup taxa Characters includedwith gaps (bp)

Number. of informative sites

Number of most parsimonious trees

Tree length

Nucleotide substitution models for Bayesian analysis (calculated with MrModeltest)

Dothiorella ITS 38 477 59 1000 135 HKY+I+GTEF1-α 37 305 1000 1000 296 GTR+GBT 27 404 51 24 80 GTR+ICombined 38 1186 240 63 517 Models from individual

gene partitionLasiodiplodia ITS* 54 483 38 154 64 GTR+I

TEF1-α* 54 293 95 52 202 HKY+GBT* 46 409 44 1000 65 GTR+GCombined 54 1185 177 48 356 Models from individual

gene partitionPseudofusicoccum ITS* 18 523 56 9 67 SYM+I+G

TEF1-α* 18 312 82 34 97 HKY+I+GCombined 18 835 138 12 165 Models from individual

gene partitionSphaeropsis ITS* 8 472 12 17 13 K80

TEF1-α* 8 317 48 2 64 HKY+GBT* 8 379 19 1 24 HKY+GCombined 8 1168 79 4 102 Models from individual

gene partition* Phylogenetic trees not shown.

TABLE 3. Polymorphic nucleotides in the ITS, TEF1-α and BT for isolates of Dothiorella tectonae, Do. brevicollis, Do. longicollis Do. striata and Do. uruguayensis.

Isolate in yellow highlight is newly taxon.


Taxonomy

Dothiorella tectonae Doilom, L.A. Shuttleworth, & K.D. Hyde, sp. nov.

Index Fungorum number: IF550706, Facesoffungi number: FoF00165 (Figure 5).

Etymology:—Species name refers to the host genus Tectona, from which the fungus was first collected. Saprobic on dead branch of Tectona grandis. Sexual morph: Undetermined. Asexual morph: Conidiomata (225–) 365–400 (–490) μm high × (170–) 270–300 (–325) μm diam. ( x = 355 × 260 μm n = 10), pycnidial, black, initially immersed, becoming erumpent through bark fissures, solitary to gregarious, uniloculate, subglobose, papillate. Papilla up to 70 μm long, 50–60 μm diam., ostiole central, periphysate. Conidiomata wall 30–105 μm thick at sides, up to 100 μm thick at base, outer layers thickened and dark, inner layer dark brown to hyaline, composed of several layers of cells of textura angularis. Conidiogenous cells (6–) 10–12 (–15) × (3.5–) 6–6.5 (–7.5) μm ( x = 10.5 × 6 μm n = 20), holoblastic, discrete, hyaline, obovoid to ellipsoidal, smooth-walled. Conidia on host (16–) 21–22 (–24) × (7.5–) 10–11 (–13) μm ( x ± S.D. = 21 ± 1.9 × 10 ± 1.2 μm, n = 50), initially hyaline and aseptate, becoming light brown to dark brown and aseptate, or 1–septate while still attached to conidiogenous cells, 1–septate at maturity, slightly constricted at the septum, oblong to ellipsoidal, ends rounded, bases obtuse, with short raised irregular striations on the surface, thick-walled, with granular content. Spermatogenous cells discrete, hyaline, smooth-walled, cylindrical, holoblastic. Spermatia 2.5–4 × 1–2 μm hyaline, aseptate, smooth, rod-shaped, with rounded ends.

FIGURE 5. Dothiorella tectonae (MFLU 14-0272, holotype) a. Conidiomata on dead branch of T. grandis. b. Section through conidioma with conidia becoming dark and aseptate or 1–septate while still attached to conidiogenous cells. c. Conidioma wall. d–e. Conidia attached to conidiogenous cells. f–j. Mature conidia (arrow showing short raised irregular striations on conidia surface). k. Spermatogenous cells and spermatia. l. Germinated mature conidium. Note d, e. stained with lactophenol cotton blue. Scale bars: a=300 μm. b=50 μm. c, e=10 μm. d, f–l=5 μm.


Culture characteristics:—Conidia germinating on PDA after 16 h. Germ tubes produced from both ends of conidia or produced laterally in some conidia. Colonies on MEA reaching 45 mm diam after 2 days in the dark at 25 °C, flattened or effuse, undulate, initially white, after 2 days becoming brownish grey (8F2) in the centre, white at edge, reaching the edge of the Petri-dish after 4 days. Cardinal temperatures for growth after four days: optimum 25–30°C, 1 mm at 5°C, 5 mm at 15°C, 8 mm at 25°C, 7 mm at 30°C. Material examined:—THAILAND, Phayao Province, Muang District, on dead branch of Tectona grandis (Lamiaceae), 12 March 2012, M. Doilom (MFLU 14-0272, holotype), ex-type culture, MFLUCC 12-0382, MUCL 55409. Notes:—Dothiorella tectonae is introduced here as a novel species based on its morphological and phylogenetic differences from known Dothiorella species. Do. tectonae differs from Do. brevicollis and Do. longicollis in having short raised irregular striations on the surfaces of mature conidia. Conidia of Do. tectonae are shorter and narrower than Do. brevicollis, but longer and wider than those of Do. longicollis (Table 4). Short raised irregular striations can also be found on conidia of Do. thailandica, however, mature conidia of Do. tectonae are longer and wider than those of Do. thailandica. Dothiorella tectonae has papillate conidiomata while Do. thailandica conidiomata are non-papillate (Table 4).

FIGURE 6. Lasiodiplodia brasiliense (MFLUCC 11-0414) a. Conidia formed on dead branches of Tectona grandis after incubation in a moist chamber for10 days. b. Colony on MEA after 1 week, and inset a conidioma on the agar surface after 1 month. c. Immature conidia attached to conidiogenous cells with paraphyses. d-f. Immature conidia attached to conidiogenous cells. g. Paraphyses. h. Immature conidia. i–k. Mature conidia. Note: c stained with lactophenol cotton blue. c–i, k. Morphology in culture. j morphology on host. Scale bars: a=100 μm. b=300 μm. c–k=10 μm.


Lasiodiplodia brasiliense M.S.B. Netto et al. in Netto et al., Fungal Diversity 67: 134 (2014)

Facesoffungi number: FoF 00628 (Figure 6).

Saprobic on dead branch of Tectona grandis. Sexual morph: Undetermined. Asexual morph: Conidiomata pycnidial formed on MEA after 1 month, dark brown, erumpent. Paraphyses up to 60 μm long, 1.5–5.5 μm wide, hyaline, septate, cylindrical, ends rounded, numerous. Conidiogenous cells 6–15 × 1.5–6 μm ( x = 10 × 3.5 μm n = 20), holoblastic, hyaline, cylindrical. Conidia on host (22–) 26–27 (–29) × 12–16 μm ( x ± S.D. = 26 ± 1.6 × 14 ± 1 μm n = 30), in culture (19–) 25–27 (–28) × 12–17 μm ( x ± S.D. = 25 ± 2 × 15 ± 1 μm n = 30), initially hyaline and aseptate, becoming 1-septate, dark brown, thick-walled, ellipsoid to obovoid, guttulate, apex broadly rounded, base truncate or round, with longitudinal striations from apex to base. Culture characteristics:—Conidia germinating on PDA after 5 h. Germ tubes produced from both ends of conidia. Colonies on MEA reaching 50 mm diameter after 1 day in the dark at 25 °C, fast growing, raised, fluffy, undulate, dense, filamentous, convex with papillate surface, initially white, after 2 days becoming pale grey and becoming dark grey (1F1) after 1 week, reaching the edge of the Petri-dish after 2 days. Material examined:—THAILAND, Chiang Rai Province, Mae Fah Luang District, on dead branches of Tectona grandis, 4 May 2011, M. Doilom (living culture MFLUCC11-0414, MUCL 55406). Notes:—Lasiodiplodia brasiliense collected in this study differs from the type species in having septate paraphyses, although this may have been overlooked in the type (Netto et al. 2014). They also differ in hosts. In this study L. brasiliense was collected from T. grandis, while the type was collected from Mangifera indica. Our collection from Tectona grandis is illustrated and described here to facilitate identification from this host.

Lasiodiplodia pseudotheobromae A.J.L. Phillips et al., Fungal Diversity 28: 8 (2008)

Facesoffungi number: FoF00166 (Figure 7).

Associated with trunk canker and branch dieback symptoms, and from dead twigs and branches of Tectona grandis. Sexual morph: Undetermined. Asexual morph: Conidiomata (330–) 360–385 (–450) μm high × 230–295 μm diam. ( x = 370 × 265 μm n = 10), pycnidial, solitary or scattered, dark brown to black, initially immersed, becoming erumpent, uniloculate, globose or subglobose, with a central ostiole. Conidiomata wall 30–50 μm wide, outer layers dark brown to black, inner layers thin-walled, pale brown to hyaline, composed of 4–6 cell layers of textura angularis. Paraphyses up to 50 μm long, 1.5–3 μm wide, hyaline, mostly aseptate, cylindrical, ends rounded, numerous. Conidiogenous cells 6–12 ×3–4 μm ( x = 8 × 3 μm n = 15), holoblastic, hyaline, cylindrical. Conidia on host (22–) 27–28.5 (–33) × 13–15 μm ( x ± S.D. = 27 ± 2.7 × 14 ± 0.5 μm n = 20), initially hyaline and aseptate, becoming 1-septate at the centre, dark brown, thick-walled, ellipsoid to obovoid, guttulate, apex broadly rounded, base truncate or rounded, with longitudinal striations from apex to base. Culture characteristics:—Conidia germinating on PDA after 5 h. Germ tubes produced from both lateral ends of the ascospore. Colonies on MEA reaching 40 mm diam after 1 day in the dark at 25 °C, cotton-like, fast growing, raised, fluffy, undulate, dense, filamentous, initially white, after 1 week becoming grey (4F1) at the edge, white in the centre, reaching the edge of the Petri-dish after 2 days. Material examined:—THAILAND, Phayao Province, Chun District, Hong Hin Sub-district, on twigs of T. grandis with dieback symptoms, 23 November 2012, M. Doilom and J. Roux (living culture MFLUCC 12-0772, MUCL 55410); Phayao Province, Muang District, on dead branch of T. grandis, 12 March 2012, Phongeun Sysouphanthong, (living culture MFLUCC 12-0294); Phayao Province, Muang District, on dead branch of T. grandis, 12 March 2012, M. Doilom (living culture MFLUCC12-0295); Chiang Rai Province, Muang District, San Sai Sub-district, Pong Sa Lee Arboretum, from basal trunk canker symptoms of T. grandis, 2 December 2012, M. Doilom and K. Jatuwong (living culture MFLUCC 12-0796, MUCL 55411); Phrae Province, Song District, Ban Rainadeaw Sub-district, on dead twig of T. grandis, 30 December 2011, M. Doilom (MFLU 14-0270, living culture MFLUCC 12-0053, MUCL 55407). Notes:—Conidia and paraphyses of L. pseudotheobromae (MFLU 14-0270) were shorter and smaller than in the holotype. The variation may be related to different hosts. The type was collected from Gmelina arborea. Our collection of Lasiodiplodia pseudotheobromae on Tectona grandis is illustrated and described here to amend the previous descriptions of L. pseudotheobromae.


FIGURE 7. Lasiodiplodia pseudotheobromae (MFLU 14-0270) a, b. Conidiomata and conidia on surface of dead twig of Tectona grandis. c. Section through conidioma. d. Conidioma wall. e. Conidia attached to conidiogenous cells with paraphyses. f, g. Immature conidia. h–j. Mature conidia in two different focal planes showing longitudinal striations. k, l. Germinated mature conidia. Note e, g stained with lacto-phenol cotton blue. Scale bars: a=300 μm. b=200 μm. c=100 μm. d, f=20 μm. e, g–l=10 μm.

Pseudofusicoccum adansoniae Pavlic et al., Mycologia 100(6): 855 (2008)

Facesoffungi number: FoF00168 (Figure 8).

Associated with leaf spots of Tectona grandis. Sexual morph: Undetermined. Asexual morph; Conidiomata (60–) 100–115 (–145) μm high × (85–) 115–125 (–145) μm diam. ( x = 100 × 115 μm n = 10), pycnidial, black, solitary or scattered, immersed to semi-immersed, globose to subglobose, uniloculate, with a central ostiole. Ostiole periphysate,


necks 30–40 μm long, 25–45 μm diam. Conidiomata wall 15–30 μm wide, outer layers dark brown to black, inner layers thin-walled, pale brown to hyaline, composed of 4–6 cell layers of textura angularis. Paraphyses not observed. Conidiogenous cells (6–) 10.5–15.5 (–18) × (3.5–) 5–6 (–8) μm ( x = 11.5 × 5.3 μm n = 15), holoblastic, cylindrical to ellipsoidal, hyaline, smooth-walled. Conidia (20–) 27.5–29.5 (–39) × 6–10 μm ( x ± S.D. = 28 ± 4 × 8 ± 1.0 μm n = 40), fusiform to ellipsoidal, hyaline, aseptate, slightly bent or irregularly shaped, smooth-walled, with fine granular content, apex broadly rounded, base rounded to truncate, covered with a persistent mucus layer. Culture characteristics:—Conidia germinating on PDA after 24 h. Germ tubes produced from the ends of the conidia. Colonies on MEA reaching 45 mm diameter after 2 days at the dark at 25 °C, initially whitened, after 3 days become greenish-grey (26F2) at the centre, white at the edge, fast growing, raise, fluffy, dense, filamentous, undulate, convex with papillate surface, reaching the edge the Petri-dish after 4 days. Material examined:—THAILAND, Chiang Rai Province, Muang District, associated with leaf spot of Tectona grandis, 17 July 2013. M. Doilom (living culture MFLUCC 13-0705, MUCL 55413, MFLUCC 14-0516 and MFLUCC14-0517). Notes:—Conidia of P. adonsoniae collected in the current study are longer and wider (28 × 8 μm versus 22.5 × 5.2 μm), than those reported by Pavlic et al. (2008) for the type. This may be due to difference substrates and lifestyle as isolates in current study were associated with leaf spots and observed directly on the host, while those from the type were isolated from asymptomatic twigs on Adansonia gregorii (Pavlic et al. 2008) with characters observed in culture on pine needles.

FIGURE 8. Pseudofusicoccum adansoniae (MFLU 15-0731) a, b. Leaf spot on T. grandis with associated conidiomata. c. Section through conidioma. d–f. Conidia attached to conidiogenous cells. g–o. Conidia. Scale bars: a=1000 μm. b=300 μm.c=30 μm. d=20 μm. e, g–o=10 μm. f=5 μm.


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Conclusions

This was a preliminary study of Botryosphaeriaceae species from T. grandis, but is the most detailed to date for this host. Six species from four different genera of Botryosphaeriaceae were found, namely Dothiorella tectonae sp. nov., Lasiodiplodia brasiliense. L. pseudotheobromae, L. theobromae, Pseudofusicoccum adansoniae and Sphaeropsis eucalypticola. All taxa are first reports on T. grandis in Thailand. Lasiodiplodia pseudotheobromae associated with trunk cankers and dieback and Pseudofusicoccum adansoniae with leaf spots. Amendments in the previous descriptions of Lasiodiplodia brasiliense, L. pseudotheobromae and P. adansoniae were provided as isolates from T. grandis varied slightly in morphological characters from the type descriptions. More detailed investigations will most likely lead to the discovery of additional species from T. grandis on teak in Thailand.

Acknowledgements

This work was financially supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program grant (No. Ph.D./0072/2553 in 4.S.M.F./53/A.2), Mae Fah Luang University grant for studying Dothideomycetes (No. 56101020032), the Tree Pathology Co-operative Programme (TPCP), Forestry and Agricultural Biotechnology Institute, University of Pretoria, and the National Research Foundation (NRF) of South Africa for funding to undertake the molecular work.

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